Holographic imaging device and method

ABSTRACT

A holographic imaging device is disclosed. In one aspect, the holographic imaging device comprises an imaging unit comprising at least two light sources, wherein the imaging unit is configured to illuminate an object by emitting at least two light beams with the at least two light sources. A first and second light beams have different wave-vectors and wavelengths. The holographic imaging device further comprises a processing unit configured to obtain at least two holograms of the object by controlling the imaging unit to sequentially illuminate the object with respectively the first light beam and the second light beam, construct at least two 2D image slices based on the at least two holograms, wherein each 2D image slice is constructed at a determined depth within the object volume, and generate a three-dimensional image of the object based on a combination of the 2D image slices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims foreign priority to EP 19204031.9, filed Oct.18, 2019, the contents of which are incorporated by reference herein intheir entirety.

BACKGROUND OF THE INVENTION Technological Field

The disclosed technology relates to a holographic imaging device and toa method for generating a three-dimensional (3D) image of an object,based on obtaining holograms of the object using at least two lightbeams. In particular, the holographic imaging device and the method mayimprove axial resolution of conventional inline holographic imaging. Theholographic imaging device and the method may be applicable formicroscopic imaging of volume samples used for quantitative imageanalysis.

Description of the Related Technology

Conventional lens-free imaging (e.g., based on inline holography) is apowerful microscopic imaging method, which does not require any lensesor other optical or mechanical components to form an image. While it hasmany advantages, for example hardware simplicity, one of its knownlimitations is its weak signal localization in 3D space, and, thus, ithas been popular for imaging planar (i.e., two dimensional (2D)) sparsesamples localized in a surface parallel to the image sensor.

Lens-free imaging on samples with a 3D structure is challenging, due totwo distinct reasons. First, this imaging method has a limited depth ofresolution, due to low “effective NA” or “missing cone in k-space” ofthe system. This limitation combined with the holographic nature of theimage recording, i.e., the samples along the beam path are recorded inthe optical image regardless of their depth, makes the imaging of 3Dsamples challenging. The signals from different depths can interferewith each other and are not localized. Second, the loss of phaseinformation by hologram recording leads to a problem of twin imagenoise. Twin images arise because the image reconstruction process yieldsidentical solutions for the object and the imaginary mirror objectlocated at the same distance to an imager but with a negative sign. Inpractice, this effect can lead to a merging of the original object imagewith its defocused image appearing together in the reconstructed image.The two effects combined create a considerable problem for applyinglens-free imaging to 3D samples.

The two effects are visually explained in the diagram 110 of FIG. 11.The diagram 110 illustrates a sample consisting of multiple 10-μm-sizedbeads randomly distributed in a volume. The hologram obtained from thesebeads is reconstructed at a certain slice in the volume. Even though thein-focus beads can be identified visually in the slice reconstruction,the signal originating from the beads from a different slice stronglyinterferes with the signal originating from the beads in the slice.Furthermore, the in-focus beads signal is corrupted with the beadssignal originating from the “ghost beads” located in a far distance.

SUMMARY OF CERTAIN INVENTIVE ASPECTS

In view of the above-mentioned disadvantages, embodiments of thedisclosed technology aim to improve axial resolution of conventionalinline holographic imaging. An objective is in particular to provide aholographic imaging device and a method, which enable 3D representationof objects. To this end, a main goal is out-of-focus plane signalsuppression in a 2D image generated by a lens-free system.

The objective is achieved by the embodiments of the disclosed technologyprovided in the enclosed independent claims. Advantageousimplementations of these embodiments are defined in the dependentclaims.

In particular, a distinguishable signature for the out-of-focus objectsmay be determined, and then the out-of-focus signals may be suppressedwhile preserving the in-focus signal.

The main advantages of the embodiments of the disclosed technology maybe summarized as follows:

-   -   Lower hardware complexity compared to, e.g., conventional        tomography methods.    -   Lower costs, higher reliability, easy to maintain and service        the device.    -   Lower computational complexity compared to        deconvolution/topography methods, which may provide faster        results and lower power consumption.

A first aspect of the disclosed technology provides a holographicimaging device comprising an imaging unit comprising at least two lightsources, wherein the imaging unit is configured to illuminate an objectby emitting at least two light beams with the at least two lightsources, wherein a first light beam has a first wave-vector and a firstwavelength and a second light beam has a second wave-vector that isdifferent from the first wave-vector and a second wavelength that isdifferent from the first wavelength; and a processing unit configured toobtain at least two holograms of the object by controlling the imagingunit to sequentially illuminate the object with the first light beam andthe second light beam, respectively, construct at least twotwo-dimensional (2D) image slices based on the at least two holograms,wherein each 2D image slice is constructed at a determined depth withinthe object volume, and generate a three-dimensional (3D) image of theobject based on a combination of the 2D image slices.

The holographic imaging device of the first aspect may be based on alens-free system, which may remove out-of-focus plane signals in theconstructed 2D image slices. In particular, the holographic imagingdevice may provide techniques to create a distinguishable signature forthe out-of-focus objects, and then remove (i.e., suppress) theout-of-focus signals while preserving the in-focus signal.

More specifically, the holographic imaging device can use the imagingunit and an illumination method entailing multiple light beams with bothan angular (wave vector) plurality and a spectral (wavelength)plurality. The holograms recorded with such pluralities may further bereconstructed digitally based on the 2D diffraction theory and imageprocessing algorithms. For example, the processing unit of theholographic imaging device may provide an algorithm, which may use thedifferences in recorded holograms generated by the angular and spectralpluralities in an iterative procedure, in which at each step thein-focus signals are identified and cleaned from the out-of-focussignals, until the algorithm converges to the desired result. Thisprocess may be repeated several times at different depths (e.g., depthswithin the object). The depth within the object may be, for example, adistance from a predefined position, e.g., front to the back of theobject, a horizontal measurement of the object's protrusion into space,a measurement perpendicular to the object's width, etc.

The holographic imaging device (e.g., the processing unit or thealgorithm provided in the processing unit) may combine the processedslices for obtaining a three-dimensional representation of the sample.

In an implementation of the holographic imaging device, the firstwave-vector and/or the second wave-vector are predetermined based onrelative positions of the at least two light sources.

In particular, the holographic imaging device may also work with lowerincidence angles, resulting in a compact (and practical forimplementation in instruments) light source as well as a larger field ofview due to lower area of “non-overlap” between illumination conescompared to the conventional topography techniques.

In some embodiments of the disclosed technology, a calibration of theholographic imaging device may be required. For example, the holographicimaging device may be sensitive to errors in the light source positions.The first wave-vector and/or the second wave-vector may be determinedand the holographic imaging device may be calibrated.

In a further implementation of the holographic imaging device, theprocessing unit is further configured to obtain at least twophase-retrieved holograms based on estimating a phase of each of the atleast two holograms. This provides the advantage that the out-of-focusghost image of the object may be removed.

In a further implementation of the holographic imaging device, the phaseof a first hologram is estimated based on performing an iterative phaseretrieval procedure by using a second hologram obtained with a differentwavelength of illumination.

In a further implementation of the holographic imaging device, theprocessing unit is further configured to determine a signature for anout-of-focus plane signal originating from a ghost image of the object,based on a comparison of the at least two phase-retrieved holograms, andremove, from the at least two phase-retrieved holograms, at least oneout-of-focus plane signal originating from the ghost image of theobject.

In particular, since the optical path between the object and, forexample, an imager of the holographic imaging device changes fordifferent illumination beams, this may result in a difference betweenthe obtained holograms. Moreover, the changes in the optical paths maybe used for determining (e.g., removing) different ghost images of thesame object in the reconstructed hologram.

In a further implementation of the holographic imaging device,constructing the at least two 2D image slices comprises obtaining, basedon the at least two phase-retrieved holograms, at least two 2D imageslices each for a respective depth within the object volume.

In a further implementation of the holographic imaging device, theprocessing unit is further configured to compare the at least two 2Dimage slices, identify spatially overlapped signals in the at least two2D image slices indicating in-focus signals of the object, and identifysubstantially shifted signals in the at least two 2D image slicesindicating out-of-focus signals originating from other real objectslocated in out-of-focus-planes.

In a further implementation of the holographic imaging device, theprocessing unit is further configured to remove, from the at least two2D image slices, at least one identified out-of-focus plane signaloriginating from another real object, and perform a noise removalprocedure for minimizing a difference between the at least two 2D imageslices. This provides the advantage that the out-of-focus real objectsignals (for example, of another real object, which may be located at adifferent depth) may be determined and may further be removed.

In a further implementation of the holographic imaging device,performing the noise removal procedure comprises removing out-of-focusplane signals that are spatially dislocated among the at least two 2Dimage slices, and/or determining and maintaining in-focus signals thatare overlapped in the at least two 2D image slices.

In a further implementation of the holographic imaging device, theprocessing unit is further configured to stack the constructed at leasttwo 2D image slices, for generating the 3D image of the object, whereineach 2D image slice is used at its determined depth within the objectvolume. This provides the advantage that the generated 3D image of theobject may be provided. Moreover, for example, the exact volumes of theobjects in the sample may be determined, which may enable quantitativeimage analysis. Such quantitative image analysis may have application involumetric imaging of biological samples, for example, bacteria, cellsor other samples in suspension, colony, reactor etc. This is important,for example, to determine the growth rate, motility or division behaviorof the cells, bacteria, etc., under different environmental conditions.

Another application of the holographic imaging device may be in airquality monitoring, water quality monitoring, pollen monitoring,industrial monitoring of fluids or gases, and in principle any imagingapplication requiring volumetric imaging capability at high lateral andaxial resolution implemented as a simple, miniature and cost-effectiveimaging setup.

In a further implementation of the holographic imaging device, theimaging unit is further comprising at least one imager, and wherein eachlight source is arranged at a predefined position from the at least oneimager.

In a further implementation of the holographic imaging device, the atleast one imager is based on a Complementary Metal Oxide Semiconductor(CMOS) image sensor.

In a further implementation of the holographic imaging device, the firstwavelength and/or the second wavelength are within a range of 300 nm to900 nm.

In particular, the first wavelength and/or the second wavelength may bewithin visible spectrum.

A second aspect of the invention provides a method for a holographicimaging device, the method comprises illuminating, by an imaging unitcomprising at least two light sources, an object by emitting at leasttwo light beams with the at least two light sources, wherein a firstlight beam has a first wave-vector and a first wavelength and a secondlight beam has a second wave-vector that is different from the firstwave-vector and a second wavelength that is different from the firstwavelength, obtaining, by a processing unit, at least two holograms ofthe object by controlling the imaging unit to sequentially illuminatethe object with respectively the first light beam and the second lightbeam, constructing, by the processing unit, at least two two-dimensional(2D) image slices based on the at least two holograms, wherein each 2Dimage slice is constructed at a determined depth within the objectvolume, and generating, by the processing unit, a three-dimensional (3D)image of the object based on a combination of the 2D image slices.

The method of the second aspect can be further developed according tothe foregoing implementations of the holographic imaging device of thefirst aspect. The method of the second aspect thus achieves the sameadvantages as the holographic imaging device of the first aspect and itsimplementations.

A third aspect of the disclosed technology provides a computer programwhich, when executed by a computer, causes the method of the secondaspect to be performed.

In some embodiments, the computer program may be provided on anon-transitory computer-readable recording medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The above described aspects and implementations are explained in thefollowing description of embodiments with respect to the encloseddrawings:

FIG. 1 shows a schematic view of a holographic imaging device forgenerating a 3D image of an object, according to an embodiment of thedisclosed technology;

FIG. 2 shows another schematic view of the holographic imaging device,according to an embodiment of the disclosed technology;

FIG. 3 shows a flow chart of a method of generating a 3D image of anobject;

FIGS. 4A-B shows different obtained holograms;

FIG. 5 shows a schematic view of a diagram illustrating obtaining aphase-retrieved hologram;

FIGS. 6A-B shows exemplarily obtained holograms with the firstwave-vector (FIG. 6A) and the second wave-vector (FIG. 6B);

FIGS. 7A-B shows an image of planar samples obtained by, a conventionaldevice (FIG. 7A) and the holographic imaging device of the invention(FIG. 7B);

FIGS. 8A-B shows an image slice from a sample including micron beadsdispersed in a 500-micron thick gel obtained by a conventional device(FIG. 8A) and the holographic imaging device of the disclosed technology(FIG. 8B);

FIGS. 9A-B shows another image slice from the sample of FIGS. 8A-Bobtained by, a conventional device (FIG. 9A) and the holographic imagingdevice of the disclosed technology (FIG. 9B);

FIG. 10 shows a method for the holographic imaging device, according toan embodiment of the disclosed technology; and

FIG. 11 shows problems of out-of-focus signals in 2D image slicesobtained by a conventional holographic imaging device.

DETAILED DESCRIPTION OF CERTAIN ILLUSTRATIVE EMBODIMENTS

FIG. 1 shows a schematic view of a holographic imaging device 10 forgenerating a 3D image of an object, according to an embodiment of thedisclosed technology.

The holographic imaging device 10 comprises an imaging unit 11 having atleast two light sources 111, 112. The imaging unit 11 is configured toilluminate an object 12 by emitting at least two light beams with the atleast two light sources 111, 112, wherein a first light beam has a firstwave-vector and a first wavelength and a second light beam has a secondwave-vector that is different from the first wave-vector and a secondwavelength that is different from the first wavelength.

The holographic imaging device 10 may thus use an illumination methodentailing multiple light beams with both angular and spectralpluralities.

The holographic imaging device 10 further comprises a processing unit 13configured to obtain at least two holograms of the object 12 bycontrolling the imaging unit 11 to sequentially illuminate the object 12with respectively the first light beam and the second light beam,construct at least two 2D image slices based on the at least twoholograms, wherein each 2D image slice is constructed at a determineddepth within the object volume, and generate a 3D image of the object 12based on a combination of the 2D image slices.

The processing unit 13 may comprise a circuitry. The circuitry maycomprise hardware (e.g., the imaging unit, a display, the processingunit (e.g., a Central Processing Unit (CPU)), a memory, etc.) andsoftware (e.g., a program for constructing 2D image slices, aprogram/algorithm for generating 3D image, etc.).

For example, the processing unit 13 may obtain the holograms. Moreover,the holograms recorded with such pluralities may be reconstructeddigitally based on the 2D diffraction theory and image processingalgorithms.

The holographic imaging device 10 (e.g., its processing unit 13, or aprogram such as an algorithm running on the processing unit 13) may usethe differences in recorded holograms generated by the angular andspectral pluralities in an iterative procedure in which at each step thein-focus signal is identified and cleaned from the out-of-focus signal.The holographic imaging device 10 (e.g., its processing unit 13) mayrepeat this procedure several times at different depths.

Moreover, the holographic imaging device 10 (e.g., its processing unit13) may combine the 2D image slices to obtain a 3D image (i.e., a 3Drepresentation of the sample).

Reference is made to FIG. 2, which is another schematic view of theholographic imaging device 10, according to an embodiment of theinvention.

The imaging unit 11 of the holographic imaging device 10 comprises threelight sources including the “light source 1” 111 illuminating with thefirst wavelength λ1, the “light source 2” 112 illuminating with thesecond wavelength λ2, the “light source 3” 211 illuminating with thethird wavelength λ3.

The holographic imaging device 10 further comprises an imager 22 and theprocessing unit 14 (e.g., a computer).

The holographic imaging device 10 of FIG. 2 configured to illuminatemultiple light beams with the wave-vector (angular) and wavelength(spectral) pluralities. For example, three light beams are generatedfrom three light sources 111, 112, 211 which are in or at differentlocations with respect to the origin of the imaging system. Furthermore,each of the light sources 111, 112, 211 emits a corresponding light beamwith a different wavelength. The pluralities of wave-vectors andwavelengths may lead (e.g., may be used) to suppressing two distinctsources of out-of-focus plane signals.

Reference is made to FIG. 3, which is a flow chart of a method 100 forgenerating a 3D image of an object. The method 100 may be performed bythe holographic imaging device 10 (of FIG. 1 and/or FIG. 2).

Without limiting the present disclosure, in the following, the method100 is discussed exemplarily being performed by the holographic imagingdevice 10.

At step S101, the holographic imaging device 10 acquires series ofholograms of object 12 illuminated sequentially with light sources 111,112, 211 and transfer the data to the computer 14.

At step S102, the holographic imaging device 10 performs iterativereconstruction of a 2D image slice at a chosen depth based on thedeveloped algorithm.

At step S103, the holographic imaging device 10 repeats thereconstruction process at different depths within the extent of theobject 12.

At step S104, the holographic imaging device 10 combines the 2D imageslices to obtain a 3D representation of the object 12.

Reference is made to FIG. 4A and FIG. 4B, in which FIG. 4A shows anexample of recorded holograms (three holograms in sequence provided as avideo) of a sample obtained by the holographic imaging device 10 withthree light sources 111, 112, 211 with different wavelengths but thesame wave-vector directions. The main image features on the hologram(the interference patterns) look very similar to each other except thatthe fine fringes on the interference pattern are observed to shift withdifferent wavelength of illumination.

The differences between the holograms are observed, since the opticalpath between the objects 12 in the sample and the imager 22 changes withdifferent illumination beams despite the fixed physical distance. Thedifference in the optical paths can result in different ghost images ofthe same object 12 in the reconstructed hologram. Thus, the plurality ofillumination wavelengths (illuminated by the holographic imaging device10) results in a distinguishable signature for the out-of-focus planesignal originating from the ghost image of the object 12.

The plurality of wavelengths in practice may be within a range of 300 nmto 900 nm (for example, it may be limited within visible spectrum λ=400nm-700 nm, in which CMOS image sensors are typically sensitive andoptical properties of objects do not vary widely). While such range issufficient to create distinguishable signatures between the object 12and its ghost in practice (typically located hundreds of microns awayfrom the object in depth), this approach may not be useful for signalsoriginating from other real objects located in out-of-focus planes thatare relatively close to the in-focus plane slice.

The holographic imaging device 10 is further configured to provide theplurality of wave-vectors of light beams to overcome the out-of-focusplane signals. FIG. 4B shows an example of recorded holograms of thesample with three light sources 111, 112, 211 with differentwave-vectors (three holograms in sequence provided as a video inFIG.4B). Differently from FIG. 4A, the features in the holograms shiftlaterally according to the wave-vector of the illumination beam. Thestrength of the relative shift between the holograms depends on thedistance of the objects 12 from the image sensor 22. The closer theobject 12 is to the imager 22, the smaller the shift with varyingwave-vector will be. The arrows in FIG. 4B indicate to three distinctfeatures in the hologram sequence. These three features originate fromobjects located three different depths. The feature in the bottom sideof the hologram hardly shifts while the other two features shift visiblyin the hologram sequence. A careful inspection suggests that theL-shaped feature shifts less than the circular one indicating that theL-shaped object is closer to the imager in distance. Thus, the pluralityof illumination wave-vectors (provided by the holographic imaging device10) results in a distinguishable signature for the signals originatingfrom objects located at different depths. The strength of thedistinguishable signature is a function of illumination beam angle andthe pixel size of the imager 22. The larger the angle of theillumination is and the smaller the imager pixel size is, the larger thesignature distinguishing two objects closely spaced slices will be.

It is important to note that the plurality of illumination wave-vectorsalone may not be useful in creating a distinguishable signature of theout-of-focus plane originating from the ghost object image, since theghost image shifts together with the object image and both alwaysoverlap with each other regardless of the wave-vector direction.Therefore, the holographic imaging device 10 provides both types ofillumination pluralities (wave-vector and wavelength) in creatingdistinguishable signatures to effectively suppress different types ofout-of-focus plane signals in the reconstructed images. A discussion ofprocessing the multiple holograms to achieve the out-of-focus signalsuppression is provided below.

In the following, description of the 2D image construction andgenerating the 3D image is provided.

Once the holographic imaging device 10 obtains the multiple holograms(e.g., acquired with the mentioned illumination method), the next stepis digitally processing them to obtain 2D image slices of the object atdifferent depths with suppressed out-of-focus signals. The steps ofimage reconstruction and processing may be split into three steps asfollows.

The goal of the first step is to identify the wave-vectors of the lightsources using a sample suitable for the purpose. This may be a practicalcalibration procedure before taking a measurement of sample. Forexample, in some embodiments, accurate knowledge of the wave-vectorsassists in the subsequent image reconstruction steps.

Reference is made to FIG. 5, which shows a schematic view of a diagram50 for determining the first wave-vector and the second wave-vector andobtaining a phase-retrieved hologram.

Initially, the holographic imaging device 10 may determine the firstwave-vector and the second wave-vector and may calibrate the position ofthe light sources 111, 112, 113 based thereon.

-   -   The goals of the first step may be:        -   Determining the relative source positions (x, y).        -   Converting the source positions into wave-vectors with a            known source displacement of z.    -   The performed operations of the first step may be summarized as        follows:        -   Estimating the relative shift of the holograms with            sub-pixel accuracy, for example, using an algorithm based on            a fast implementation of cross correlation between the            images.        -   Reconstructing firstly the holograms in focus with an            “initial guess of the positions”.        -   Using the reconstructed object images for determining the            sharp features and obtaining a better accuracy for            registration.        -   Applying an image registration procedure for the            reconstructed images and determining the error in the            initial guess.        -   Identifying accurate x, y positions per source.        -   Determining the wave-vectors.

In the second step, the holographic imaging device 10 may further obtainthe phase-retrieved holograms.

For instance, the holograms acquired with the imager 22 may contain onlythe intensity of the hologram and the phase component may be lost. Theholographic imaging device 10 may apply a phase retrieval procedure toestimate the phase of holograms using the plurality of the wavelengthsof illumination. It is important to note that the second phase isrepeated for all the holograms acquired from the imager. Therefore, aphase estimation is done on all the holograms. The output of the secondstep is the three phase-retrieved holograms, which will be passed tostep three.

-   -   The goals of the second step may be:        -   Phase retrieval for the holograms using the wavelength            plurality.        -   Removal of an out of focus plane ghost image signal.    -   The performed operations may be summarized as follow:        -   Phase retrieval may be based on propagating complex valued            holograms and replacing the phases at even steps (steps of a            single iteration drawn for H1 on the left).        -   Choosing a slice (z_(obj)) in the object volume for            reconstruction.        -   After N iterations, repeating step one for obtaining            ghost-free image of the object with wavevector-1.        -   Repeating the process for all the holograms (e.g., H2 and            H3).        -   Obtaining as final output, three holograms with retrieved            phases.

The reconstruction of these holograms in the next step may result in acleaned ghost image signal.

At the third step, the object image reconstruction may be performed byusing the phase retrieved holograms corresponding to three differentwave-vector illumination beams. The first process is to reconstructthree 2D image slices within the object volume from the phase retrievedholograms. For example, the holographic imaging device 10 may use a 2Ddiffraction theory based on field propagation such as angular spectrum,etc. Note that the reconstructed slices are free-from out-of-focussignals originating from the ghost image but not the signals generatedfrom the objects (for example, another object than the desired object)located near the slice depth. An example of such slices is shown in FIG.6A and FIG. 6B. These two images reveal that in-focus signals overlapspatially in two images while the out-of-focus signals are shiftedrelatively.

Once the reconstructed slices are obtained, the holographic imagingdevice 10 may suppress the out-of-focus signals using the threereconstructed slices and a noise removal algorithm designed to minimizedifferences between the reconstructed slices from three differentholograms. The noise removal algorithm succeeds to reject out-of-focusplane signal since only the in-focus signal overlaps in all differentslices while out-of-focus signal is spatially dislocated among the threeslices.

The holographic imaging device 10 may use different noise removalprocedures. For example, the simplest approach may be averaging thethree reconstructed slices as the overlapping in-focus signal will beenhanced while non-overlapping out-of-focus signal will be averaged out.More advanced approaches may be used to suppress the unwanted signals.One of such approaches is formulating the in-focus signal as an inverseproblem and minimizing the error using regularization in an iterativeoptimization algorithm. L1, L2 and total variation (TV) normalizationmay be used in image processing to solve inverse problems depending onthe nature of the problem. Such an approach can be used to minimize theerror occurring in three distinct reconstructed slices enabled by thedifferent wave-vectors.

The holographic imaging device 10 may process the slices at other depthsin a similar procedure as explained above. Once all the slices in thesample volume are processed, the slices can be stacked into a 3D datarepresentation. The holographic imaging device 10 may enable enhanceddepth resolution to create true 3D imaging of complex samples.

Experimental validation of the proposed invention is done, and someinitial results (obtained by the holographic imaging device 10) arepresented in the following, without limiting the present disclosure.

FIGS. 7A-B shows an image of planar samples of metal lines on asubstrate obtained by, a conventional device (FIG. 7A) and theholographic imaging device 10 of the invention (FIG. 7B).

As it can be derived from FIG. 7B, the holographic imaging device 10removes the ghost image related out-of-focus signal.

FIGS. 8A-B shows an image slice (slice #1) from a sample includingmicron beads dispersed in 500-micron thick gel obtained by, aconventional device (FIG. 8A) and the holographic imaging device of theinvention (FIG. 8B).

As it can be derived from FIG. 8B, the holographic imaging device 10removes (from slice #1) the out-of-focus plane signals.

FIGS. 9A-B shows another image slice (slice #2) from the sample of FIGS.8A-B obtained by, a conventional device (FIG. 9A) and the holographicimaging device of the invention (FIG. 9B).

As it can be derived from FIG. 9B, the holographic imaging device 10removes (from slice #2) the out-of-focus plane signals.

As it can be derived from the two image slices (e.g., FIG. 8B and FIG.9B) that are obtained from the sample containing 1-micron sized beadsdistributed in a 3D volume, the holographic imaging device 10effectively suppresses the out-of-focus signal originating from beads inother depths.

FIG. 10 shows a method 200 according to an embodiment of the inventionfor a holographic imaging device 10. The method 200 may be carried outby the holographic imaging device 10, as it is described above.

The method 200 comprises a step S201 of illuminating, by an imaging unit11 comprising at least two light sources 111, 112, an object 12 byemitting at least two light beams with the at least two light sources,wherein a first light beam has a first wave-vector and a firstwavelength and a second light beam has a second wave-vector that isdifferent from the first wave-vector and a second wavelength that isdifferent from the first wavelength.

The method 200 further comprises a step S202 of obtaining, by aprocessing unit 13, at least two holograms of the object 12 bycontrolling the imaging unit 11 to sequentially illuminate the object 12with respectively the first light beam and the second light beam.

The method 200 further comprises a step S203 of constructing, by theprocessing unit 13, at least two 2D image slices based on the at leasttwo holograms, wherein each 2D image slice is constructed at adetermined depth within the object volume.

The method 200 further comprises a step S204 of generating, by theprocessing unit 13, a 3D image of the object 12 based on a combinationof the 2D image slices.

What is claimed is:
 1. A holographic imaging device comprising: animaging unit comprising at least two light sources and configured to:illuminate an object by emitting a first light beam with a first one ofthe at least two light sources and a second light beam with a second oneof the at least two light sources, wherein the first light beam has afirst wave-vector and a first wavelength and the second light beam has asecond wave-vector that is different from the first wave-vector and asecond wavelength that is different from the first wavelength; and aprocessing unit configured to: obtain at least two holograms of theobject by controlling the imaging unit to sequentially illuminate theobject with respectively the first light beam and the second light beam,obtain at least two phase-retrieved holograms based on estimating aphase of each of the at least two holograms, processing the at least twophase-retrieved holograms by: determining a signature for anout-of-focus plane signal originating from a ghost image of the object,based on a comparison of the at least two phase-retrieved holograms, andremoving, from the at least two phase-retrieved holograms, at least oneout-of-focus plane signal originating from the ghost image of theobject, construct at least two two-dimensional (2D) image slices basedon the at least two processed phase-retrieved holograms, wherein each 2Dimage slice is constructed at a determined depth within an object volumeof the object, and generate a three-dimensional (3D) image of the objectbased on a combination of the 2D image slices.
 2. The holographicimaging device according to claim 1, wherein: at least one of the firstwave-vector or the second wave-vector are predetermined based onrelative positions of the at least two light sources.
 3. The holographicimaging device according to claim 1, wherein: the phase of a firsthologram is estimated based on performing an iterative phase retrievalprocedure by using a second hologram obtained with a differentwavelength of illumination.
 4. The holographic imaging device accordingclaim 1, wherein: constructing the at least two 2D image slicescomprises obtaining, based on the at least two processed phase-retrievedholograms, at least two 2D image slices each for a respective depthwithin the object volume.
 5. The holographic imaging device according toclaim 4, wherein the processing unit is further configured to: comparethe at least two 2D image slices, identify spatially overlapped signalsin the at least two 2D image slices indicating in-focus signals of theobject, and identify substantially shifted signals in the at least two2D image slices indicating out-of-focus signals originating from otherreal objects located in out-of-focus-planes.
 6. The holographic imagingdevice according to claim 5, wherein the processing unit is furtherconfigured to: remove, from the at least two 2D image slices, at leastone identified out-of-focus plane signal originating from another realobject, and perform a noise removal procedure for minimizing adifference between the at least two 2D image slices.
 7. The holographicimaging device according to claim 6, wherein performing the noiseremoval procedure comprises at least one of: removing out-of-focus planesignals that are spatially dislocated among the at least two 2D imageslices, or determining and maintaining in-focus signals that areoverlapped in the at least two 2D image slices.
 8. The holographicimaging device according to claim 6, wherein the processing unit isfurther configured to: stack the constructed at least two 2D imageslices, for generating the 3D image of the object, wherein each 2D imageslice is used at its determined depth within the object volume.
 9. Theholographic imaging device according to claim 1, wherein the imagingunit is further comprising at least one imager and wherein each lightsource is arranged at a predefined position from the at least oneimager.
 10. The holographic imaging device according to claim 9, whereinthe at least one imager is based on a complementary metal oxidesemiconductor (CMOS) image sensor.
 11. The holographic imaging deviceaccording to claim 1, wherein: at least one of the first wavelength orthe second wavelength are within a range of 300 nm to 900 nm.
 12. Amethod of using a holographic imaging device, the method comprises:illuminating, by an imaging unit comprising at least two light sources,an object by emitting a first light beam with a first of the at leasttwo light sources and a second light beam with a second of the at leasttwo light sources, wherein a first light beam has a first wave-vectorand a first wavelength and a second light beam has a second wave-vectorthat is different from the first wave-vector and a second wavelengththat is different from the first wavelength, obtaining, by a processingunit, at least two holograms of the object by controlling the imagingunit to sequentially illuminate the object with respectively the firstlight beam and the second light beam, obtaining, by the processing unit,at least two phase-retrieved holograms based on estimating a phase ofeach of the at least two holograms, processing, by the processing unit,the at least two phase-retrieved holograms by: determining a signaturefor an out-of-focus plane signal originating from a ghost image of theobject, based on a comparison of the at least two phase-retrievedholograms, and removing, from the at least two phase-retrievedholograms, at least one out-of-focus plane signal originating from theghost image of the object, constructing, by the processing unit, atleast two two-dimensional (2D) image slices based on the at least twoprocessed phase-retrieved holograms, wherein each 2D image slice isconstructed at a determined depth within an object volume of the object,and generating, by the processing unit, a three-dimensional (3D) imageof the object based on a combination of the 2D image slices.
 13. Anon-transitory computer readable medium storing a computer programwhich, when executed by a computer, causes the method of claim 12 to beperformed.